The present invention concerns the modulation of transfer of macromolecules from the cell cytoplasm to the cell nucleus, to backbone cyclic peptides which inhibit nuclear import and to compositions and methods using these peptides to inhibit nuclear localization and virus production.
The life cycle of eukaryotic cells depends upon translocation of functional proteins from the cytoplasm into the cell nucleus in which DNA replication and RNA biosynthesis occurs. Also, nucleo-cytoplasmic transport of viral genomes is essential for the replication and assembly of many animal viruses. For example, the nuclear import of Human immunodeficiency virus (HIV-1), and of Herpes simples virus (HSV), is crucial for the productive infection of non-dividing cells (Von-Schwedler, et al.(1994), Proc.Natl.Acad.Sci. USA 91, 6992-6996), in which the nuclear membrane is intact.
The Nucear Localization Signal (NLS) is a transport signal within proteins which mediates their nuclear uptake by a complex mechanism (Melchior (1995) Curr.Opin.Cell.Biol. 7, 310-318 Garcia-Bustos, et al. (1991) Biochim. Biophys. acta 1071, 83-101). It has a semi-consensus sequence generally composed of 5-15 amino acid residues, of which a significant proportion are positively charged (lysine and arginine) (Dingwall, and Laskey (1991) TIBS 16, 478-481). Nuclear import is initiated in the cytoplasm by specific binding of the NLS to a cytoplasmic receptor, generally designated as an NLS-binding protein (NBP). A number of proteins that interact with NLS sequences nave been detected, identifying them as nuclear import receptors (Nigg, (1997) Nature 386, 779-787). The first nuclear import pathway that was discovered operates with the karyopherin heterodimer also known as importin (Gorlich et. al. (1995) Nature 377, 246-248).
Viral transport into the nucleus is made possible as their genomes are complexed with viral proteins that harbor unique NLS""s crucial for nuclear transport. At least three HIV-1 proteins are involved in nuclear import of the viral preintegration complex (PIC), thus displaying partially redundant nuclear localization activity: The HIV-1 MA, a nucleocapsid component (Bukrinsky et. al. ibid), the auxiliary protein Vpr (Heinzinger et. al. (1994) Proc. Natl. Acad. Sci. USA 91, 73 11-7315), and the viral integrase (Gallay et. al. (1997) Proc. Natl. Acad.Sci. USA 94, 9825-9830). HIV-1 also expresses several auxiliary proteins of molecular weight under 30 kDa (Miller and Sarver, (1997) Nature Medicine 3, 389-394). Out of these proteins Tat, Rev, Nef and Vif contain NLS-like sequences and accumulate, under certain conditions, within the nuclei of infected cells. It has recently been reported that Vpr (Karni et. al.(1998) FEBS Lett. 429, 421-425), Tat (Efthymiadis et. al. (1998) J Biol Chem 273, 1623-1628) and Rev (Henderson and Percipalle, (1997) J Mol Biol 274, 693-707) promote nuclear import by a distinct non-importin pathway. Linear peptides derived from NLS sequences function as an active NLS since their covalent conjugation to BSA caused nuclear import of the resulting conjugate (Bukrinsky, et al. ibid; Goldfarb, et al. (1986) Nature 322, 641-644). Such peptides also inhibit nuclear import as was demonstrated by inhibition of the HIV-1 matrix protein (MA) and its preintegration complex (Gulizia, et al. (1994) J. Virol. 68, 2021-2025). However, due to their structural flexibility and metabolic instability, the therapeutic use of linear NLS peptides as anti viral drugs is impractical.
Proteinomimetics are small molecules that mimic the structure and/or the activity of a large parent protein. The availability of such small molecules can be useful for the detailed study of the biological function, molecular structure and folding of proteins. Moreover, proteinomimetics are excellent candidates for becoming a novel type of drugs, since they overcome some of the limitations that currently hamper the therapeutic use of proteins and polypeptides such as antigenicity, metabolic instability and poor bioavailability. While many structural proteinomimetics have already been described, most of them were deprived of the biological function which characterized the parent protein. Also attempts to obtain small peptides which mimic catalytic sites of enzymes and preserve their enzymatic activity have so far failed (Corey, and Corey (1996) Proc.Natl.Acad.Sci. USA 93, 11428-11434). Very few examples of structural proteinomimetics which retain the biological activity and resemble the structure of the corresponding proteins have so far been disclosed, such as the zinc-finger (Struthers, et al. (1996) Science 271, 342-345) and the metal-binding proteinomimetics (Robertson, et al. (1994) Nature 368, 425-432; Pessi, et al. (1993) Nature 362, 367-369). We now disclose a general approach for the design and synthesis of small backbone cyclic peptides which mimic structure and the function of active regions in proteins.
Backbone cyclization is a general method by which a conformational constraint is imposed on peptides through the connection of the Nxcex1 or Cxcex1 atoms in the peptide backbone to each other or to side chains or to the carboxyl and amino termini (Gilon et al. (1991) Biopolymers 31, 745-750). Backbone cyclization has been previously shown to convert peptides into selective and metabolically-stable peptidomimetics with enhanced biological activity as compared to the linear parent peptide as discussed for instance in EPO 564,739 A2 and WO 95/33765; Byk, et al. (1996) J. Med. Chem. 39, 3174; Bitan, et al. (1997) J. Pept. Res. 49, 421). Cycloscan is a selection method based on conformationally constrained backbone cyclic peptide libraries that allows rapid detection of the most active backbone cyclic peptide derived from a given sequence as disclosed in WO 97/09344. The diversity of cycloscan, which includes modes of backbone cyclization, ring position, ring size and ring chemistry allows the Generation of a large number of sequentially biased peptides that differ solely by their conformation in a gradual discrete manner. The principles of the xe2x80x9cbackbone cyclic proteinomimeticxe2x80x9d approach are based on the following, steps: (i) elucidation of the active residues in the target protein (ii) design and modeling of an ensemble of prototypic backbone cyclic peptides that encompass the active residues and their conformation resemble that of the parent protein (iii) cycloscan of each backbone cyclic prototype until a lead compound is discovered (iv) structural analysis of the best lead and (v) optimization through iteration.
It is one object of the present invention to provide backbone cyclic peptide analogs which mimic Nuclear Localization Signal (NLS) regions in macromolecules. It is another object of the invention to utilize the backbone cyclic peptide analogs to inhibit nuclear import of NLS containing macromolecules into the cell nucleus. It is yet another object of the invention to provide backbone cyclic peptide analogs which mimic the NLS region of various proteins of the human immunodeficiency virus. Yet another object of the present invention to provide compositions and methods of using said backbone cyclic peptides to inhibit virus replication in infected cells. According to the principles of the present invention it is now disclosed that using the backbone cyclic proteinomimetic approach it is possible to design libraries of backbone cyclic peptides that mimic the region of the NLS sequence. These libraries can be used to identify molecules which can then be further optimized and refined to mimic the specific NLS sequences of particular macromolecules as required. These principles are exemplified using the NLS sequence of certain viral proteins. The backbone cyclic peptides obtained are useful to inhibit the translocation of macromolecules from the cell cytoplasm to the cell nucleus thereby disrupting the propagation of tile virus.
Currently more preferred embodiments according to the present invention include backbone cyclic peptide analogs which can mimic the action of NLS sequences and of NLS-like sequences of HIV-1. These preferred embodiments encompass backbone cyclic analogs of the NLS sequence selected from the HIV-1 viral proteins MA, Tat and Vpr.
Yet another preferred embodiment according of the present invention provides backbone cyclic analogs of a peptide within the HIV-1 protein Vif. These backbone cyclic peptides comprise analogs of residues 88 through 98 of Vif, having a short sequence of basic residues. This viral sequence inhibits but does not mediate nuclear import.
One currently most preferred embodiment of the present invention, is a backbone cyclic peptide analog of the general Formula I: 
wherein n denotes an integer from 1 to 10 or may be zero, with the proviso that when n is zero NH is also absent; m denotes an integer from 1 to 10; R denotes lysine or glutamine; AA and AA2 independently denote an amino acid residue which may be the same or different.
A more preferred embodiment according to the present invention, is a backbone cyclic peptide analog of the general Formula II: 
wherein n is an integer from 1 to 10 or may be zero, with the proviso that when n is zero NH is also absent; m is an integer from 1 to 10, and R denotes Lys or Gln.
A yet more preferred embodiment according to the present invention, is a backbone cyclic peptide analog of the general formula III: 
wherein R denotes Lys or Gln, AA denotes an amino acid residue selected from the group consisting of serine, leucine or methionine, and AA2 denotes an amino acid residue selected from the group consisting of valine, glycine or cysteine.
A currently most preferred embodiment according to the present invention is the peptide of Formula III wherein R is Lysine, AA is Leucine and AA2 is Valine which is designated herein as BCvir.
Yet another most preferred embodiment according to the present invention comprises backbone cyclic peptide analogs of the general formula IV: 
wherein m and n are each independently an integer from 1 to 10.
Pharmaceutical compositions comprising, as an active ingredient a backbone cyclic peptide capable of mimicking the NLS sequence are disclosed and claimed.
Further disclosed are methods of inhibiting the translocation of macromolecules from the cell nucleus to the cell cytoplasm by contacting the cells with an effective amount of a backbone cyclized peptide which mimics the NLS sequence of said macromolecule. These are useful for the prevention or treatment of viral infection both in vitro or in vivo in an animal in need of such treatment. The utility of these methods is exemplified by inhibiting the replication of virus in virus infected cells.